Assay to identify estrogen receptor dependent ligands that regulate the hepatic lipase promoter

ABSTRACT

The present invention relates to an assay system and method for testing compounds for their ability to regulate the hepatic lipase (HL) promoter. In particular, the invention relates to the identification of estrogen receptor ligands having this activity. Compounds that inhibit HL promoter activity are useful as leads, or on their own, to develop therapeutics in the prevention of heart disease

PRIORITY

This application is a continuation of U.S. application Ser. No.09/924,944 entitled “Assay to Identify Estrogen Receptor DependentLigands That Regulate the Hepatic Lipase Promoter, filed Aug. 8, 2001,which in turn claims priority under 35 U.S.C.§119(e) from ProvisionalPatent Application Nos. 60/223,647 and 60/255,837 filed on Aug. 8, 2000and Dec. 15, 2000, respectively. Each of the above-referencedapplications is incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to an assay system and method for testingcompounds for their ability to regulate the hepatic lipase (HL)promoter. In particular, the invention relates to the identification ofestrogen receptor ligands having this activity. Compounds that inhibitHL promoter activity are useful as leads, or on their own, to developtherapeutics in the prevention of heart disease.

BACKGROUND OF THE INVENTION

The physiological response to steroid hormones is mediated by specificinteraction of steroids with nuclear receptors, which areligand-activated transcription factors that regulate the expression oftarget genes. These receptors consist (in anaminoterminal-to-carboxyterminal direction) of a hypervariableaminoterminal domain that contributes to the transactivation function; ahighly conserved DNA-binding domain responsible for receptordimerization and specific DNA binding; and a carboxyterminal domaininvolved in ligand-binding, nuclear localization, and ligand-dependenttransactivation.

In vivo, estrogen has been identified as having utility in treatingadverse behavioral/clinical symptoms that accompany fluctuations inhormones associated with menopause in aging women, although thebiochemical basis for these effects has never been determined. As such,the treatment of behavioral effects with estrogen in human subjects hasbeen restricted to the treatment of menopause in women who demonstratesigns of deficiency in estrogen, and use in prevention of the sequelaeof menopause, namely hot flashes and osteoporosis, which are typicallycorrected by replacement therapy of estrogen.

Recently, cDNA was cloned from rat prostate and shown to havesignificant homology to a previously isolated rat estrogen receptor (ER)(Kuiper et al., Proc. Natl. Acad. Sci. USA 93:5925, 1996); this receptorwas designated ERβ to distinguish it from the previously clonedreceptor, ERα. Rat ERβ was shown to be expressed in prostate, testes,ovary, and thymus, in contrast to ERα, which is most highly expressed inuterus, breast, liver, and pituitary. A human ERβ homologue has beenreported (Mosselman et al., FEBS Letts. 392:49, 1996), having theaminoterminal sequence Gly-Tyr-Ser. A human ERβ has been described inPCT Publication WO 99/07847.

Hepatic lipase (HL) is a lipolytic enzyme that is synthesized primarilyin the liver. HL hydrolyzes triglycerides and phospholipids present inchylomicron remnants, intermediate density lipoprotein (IDL), andhigh-density lipoprotein (HDL). Through its function as a lypolyticenzyme, HL plays a major role in the metabolism of circulating plasmalipoproteins resulting in elevation of small, dense atherogenic LDL witha decrease in HDL plasma levels. Several lines of evidence demonstratethe important role of HL in HDL metabolism. Patients with a geneticdeficiency of HL have increased plasma levels of HDL cholesterol andphospholipids (Breckenridge et al., Atherosclerosis, 45:161, 1982).Increased HDL is also a hallmark of HL-deficient states induced byinfusion of anti-HL antibodies (Goldberg et al., J. Clin. Invest.70:1184, 1982), genetic manipulation (Homanics et al. J. Biol. Chem.270:2974, 1995) or naturally present in various animal models (Clay etal. Biochim. Biophys. Acta. 1002:173, 1989). Conversely, overexpressionof HL decreases plasma HDL concentrations in transgenic mice (Busch etal. J. Biol. Chem. 269:16376, 1994) and rabbits (Fan et al. PNAS91:8724, 1994).

In terms of ERT in postmenopausal women, it is well established thattrafficking of lipoprotein cholesterol is enhanced via the reversecholesterol transport system. HL activity has been shown to be reducedby 31% due to ERT, levels similar to that found in premenopausal women(Tikkanen et al. Acta Obstet Gynecol Scand Suppl. 88:83, 1979).Diminishment of HL activity by ERT is thought to improve the reversecholesterol transport system by blocking the metabolism of HDL therebymaintaining higher plasma levels of HDL. In addition, inhibition of HLactivity may result in a reduction in the amount of small, denseatherogenic LDL.

Thus, there is a need in the art to identify compounds that can modulateHL production. There is a further need in the art to identify compoundsthat can act through the estrogen receptor to modulate, and preferablyinhibit, HL expression by acting on the HL promoter.

SUMMARY OF THE INVENTION

The present invention provides for a transformed cell that expresses afunctional estrogen receptor, a C/EBP transcription factor, and areporter gene that is associated with HL. In one specific embodiment,the estrogen receptor is a human estrogen receptor. In another specificembodiment, the transcription factor is C/EBPα. The another specificembodiment, the reporter gene is luciferase.

One specific embodiment provides that the cell is a hepatocarcinomacell. In a further embodiment, the hepatocarcinoma cell is a HEPG2 cell.

The present invention also provides for an assay system for estrogenligands that modulate HL activity in a population of the transformedcells that are described above. In one specific embodiment, the estrogenreceptor is a human estrogen receptor. In another specific embodiment,the transcription factor is C/EBPα. The another specific embodiment, thereporter gene is luciferase.

One specific embodiment provides that the cell is a hepatocarcinomacell. In a further embodiment, the hepatocarcinoma cell is a HEPG2 cell.

The present invention also provides for a method of identifying acompound that regulates HL promoter activity through an estrogenreceptor that is expressed in the transformed cells that are describedabove, where a change in the level of the expression of the reportergene indicates that the test compound regulates HL activity through theexpressed estrogen receptor. The invention also contemplates a methodwhere reporter gene expression is decreased after contact of the testcompound with the assay system. In one embodiment, the test compound maybe an estrogen or estrogen analog. In specific embodiments, the presentinvention also contemplates that the estrogen receptor is a humanestrogen receptor, that the transcription factor is C/EBPα, and that HLpromoter is positioned proximal to the 5′ end of the HL coding region.Another specific embodiment also contemplates that the reporter gene isluciferase.

One specific embodiment provides that the cell is a hepatocarcinomacell. In a further embodiment, the hepatocarcinoma cell is a HEPG2 cell.

In one embodiment, the estrogen receptor, transcription factor, andreporter gene are expressed by separate vectors within the transformedcell.

DETAILED DESCRIPTION OF THE INVENTION

The present invention advantageously provides a transfection (ortransduction) screening assay for identifying compounds that regulatehepatic lipase (HL) promoter activity through the estrogen receptor. Theassay system of the invention is suitable for high throughput screening,e.g., screening thousands of compounds per assay.

The HL promoter assay can be used to identify compounds that interactwith the ER to regulate HL promoter activity of a luciferase reporterconstruct. Compounds that inhibit HL promoter activity may result inreduced HL protein production and may be useful as novel therapeutics inthe prevention of heart disease. HL is involved in lipoproteinmetabolism and reduction of its activity has been associated withelevation of plasma high density lipoprotein (HDL) levels and a decreasein small, dense LDL particles which are thought to be anti-atherogenic.

The present invention is based, in part, on construction of human liverhepatoma cells, HepG2, cotransfected with (i) a human ER expressionvector, (ii) a CAAT enhancer binding protein (C/EBPα) expression vector,and (iii) a luciferase reporter vector in which luciferase reporter geneexpression is controlled by the human HL promoter region (−1557/+43).Alternatively, (i) human ER, (ii) aC/EBPα expression vector, and (iii) aluciferase reporter vector in which luciferase reporter gene expressionis controlled by the human HL promoter region are present in one vector.This vector then may be used to transform the cell.

Transfected cells in multi-well plates were treated with estrogen ortest compounds for 16-24 hr and luciferase activity was determined incell lysates. Under these conditions, 17β-estradiol inhibited HLpromoter activity by approximately 75% after overnight treatment, inHepG2 cells transected with human ERα vectors. Repression of HL promoteractivity is both ER and ligand dependent. Thus, the assay of theinvention are applicable to other nuclear hormone receptors, includingERP receptors.

C/EBPα is a liver-enriched transcription factor which belongs to afamily of receptors called CAAT enhancer binding proteins, whichincludes C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, and C/EBPε. It induces a moredifferentiated phenotype in the HepG2 cells and activation of HLpromoter activity by any one of these family members is also regulatedby binding of an appropriate ligand to estrogen receptor.

As used herein the term “transformed cell” refers to a modified hostcell that expresses (i) a functional estrogen receptor expressed from avector encoding the estrogen receptor; (ii) a C/EBP transcription factorthat acts on a HL promoter expressed from a vector encoding thetranscription factor; and (iii) a reporter gene operatively associatedwith an HL promoter. Any host cell can be used, preferably a hepatocytecell, and more preferably a hepatocarcinoma cell. In a specificembodiment, the cell is a HepG2 cell.

A “functional estrogen receptor” is a receptor that binds estrogen orestrogen analogs and transduces a signal upon such binding. Preferablythe ER is a human ER (hER), for example hERα or HERβ. (Kioke et al. NAR15:2499, 1987; White et al. Mol. Endo. 1:735, 1987; Kuiper et al. PNAS93:5925, 1996; Tremblay et al. Mol Endo 11:353, 1997; Mosselman et al.FEBS Lett 392:49, 1996).

A “C/EBP transcription factor” is a liver-enriched transcription factorwhich belongs to a family of receptors (C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ,C/EBPε). In a specific embodiment, the transcription factor is C/EBPα.CAAT/enhancer-binding protein (C/EBP) is a transcription factorexpressed primarily in liver, fat and intestinal tissues that belongs tothe basic region-leucine zipper class (Birkenmeier et al. Genes & Dev.3:1146, 1989; Landschulz et al. Science 243:1681, 1988). Overexpressionof C/EBP in cotransfection assays stimulates transcription through C/EBPbinding sites found in promoters of target genes and suggests that it isinvolved in cell type-specific expression of genes in liver, fat andpossibly additional tissues.

The orphan nuclear receptor COUP-TF family members (ARP-1, EAR-2 &EAR-3) will also work however, they do not induce HL reporter activityas efficiently as CEBPα.

A “hepatic lipase” is involved in lipoprotein metabolism. The hepaticlipase promoter is the region upstream, e.g., about 1600 base pairsupstream, of the hepatic lipase coding region on the chromosome thatregulates expression of the protein. HL promoter activity is regulatedby ER. In a specific embodiment, the HL promoter is positioned proximalto the 5′ end of the human HL coding region. In a specific embodiment,HL promoter is the human HL promoter region from −1557 to +43, relativeto the HL coding region start site (0).

A “reporter gene” as used herein is a gene that encodes a detectableprotein. Generally, the protein is an enzyme, and can be detected bydetecting the enzymatic reaction mediated by the protein, e.g.,development of a chromogenic product or light. For example, the reportergene can encode a protein selected from the group consisting ofluciferase, green fluorescent protein, yellow fluorescent protein,β-galactosidase, chloramphenicol transferase, horseradish peroxidase,and alkaline phosphatase. Alternatively, a reporter gene encodes aprotein that can be detected, e.g., in an immunoassay. In a preferredembodiment, the protein encoded by the reporter gene is luminescent(fluorescent or phosphorescent). In a specific embodiment, the proteinis luciferase.

The cells of the invention are particularly suitable for an assay systemfor estrogen receptor ligands that modulate HL promoter activity. An“assay system” is one or more collections of cells, e.g., in a microwellplate or some other culture system. To permit evaluation of the effectsof a test compound on the cells, the number of cells in a single assaysystem is sufficient to express a detectable amount of the proteinencoded by the reporter gene under conditions of maximum reporter geneexpression. For example, in the absence of an estrogen that suppressesHL promoter activity, as exemplified herein by 17β-estradiol, thereporter gene expresses a detectable level of protein, such that areduction in the level of reporter gene expression is detectable.

A “test compound” is any molecule, such as an estrogen compound, thatcan be tested for its ability to modulate HL promoter activity throughthe ER, as set forth herein.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, and more preferably within 5% of agiven value or range. Alternatively, particularly in the measurement ofbiological processing, the term “about” or “approximately” means withinan order of magnitude, preferably within a factor of 2, of a givenvalue, e.g., a concentration of a compound that causes a half-maximalbiological effect. Thus, the term “about” or “approximately” means thata value can fall within a scientifically acceptable error range for thattype of value, which will depend on how quantitative a measurement canbe given the available tools.

As used herein, the term “isolated” means that the referenced materialis free of components found in the natural environment in which thematerial is normally found. In particular, isolated biological materialis free of cellular components. In the case of nucleic acid molecules,an isolated nucleic acid includes a PCR product, an isolated mRNA, acDNA, or a restriction fragment. In another embodiment, an isolatednucleic acid is preferably excised from the chromosome in which it maybe found, and more preferably is no longer joined to non-regulatory,non-coding regions, or to other genes, located upstream or downstream ofthe gene contained by the isolated nucleic acid molecule when found inthe chromosome. In yet another embodiment, the isolated nucleic acidlacks one or more introns. Isolated nucleic acid molecules can beinserted into plasmids, cosmids, artificial chromosomes, and the like.Thus, in a specific embodiment, a recombinant nucleic acid is anisolated nucleic acid. An isolated protein may be associated with otherproteins or nucleic acids, or both, with which it associates in thecell, or with cellular membranes, if it is a membrane-associatedprotein. An isolated organelle, cell, or tissue is removed from theanatomical site in which it is found in an organism. An isolatedmaterial may be, but need not be, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate unrelated materials,i.e., contaminants. For example, a purified protein is preferablysubstantially free of other proteins or nucleic acids with which it isassociated in a cell; a purified nucleic acid molecule is preferablysubstantially free of proteins or other unrelated nucleic acid moleculeswith which it can be found within a cell. As used herein, the term“substantially free” is used operationally, in the context of analyticaltesting of the material. Preferably, purified material substantiallyfree of contaminants is at least 50% pure; more preferably, at least 90%pure, and more preferably still at least 99% pure. Purity can beevaluated by chromatography, gel electrophoresis, immunoassay,composition analysis, biological assay, and other methods known in theart.

The use of italics indicates a nucleic acid molecule (e.g., ER cDNA,gene, etc.); normal text indicates the polypeptide or protein.

Engineered ER/HL Promoter Systems

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a recombinant nucleic acid construct, such as plasmid,phage genome, virus genome, cosmid, or artificial chromosome, to whichanother DNA segment may be attached. In a specific embodiment, thevector may bring about the replication of the attached segment, e.g., inthe case of a cloning vector. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., it is capable of replication under its owncontrol.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA is expressedand effects a function or phenotype on the cell in which it isexpressed.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, the vectors for expression of the estrogen receptor,transcription factor, and reporter gene operatively associated with theHL promoter is heterologous to a host cell in which it is expressed,e.g., a hepatocarcinoma cell.

A “gene” is used herein to refer to a portion of a DNA molecule thatincludes a polypeptide coding sequence operatively associated withexpression control sequences. In one embodiment, a gene can be a genomicor partial genomic sequence, in that it contains one or more introns. Inanother embodiment, the term gene refers to a cDNA molecule (i.e., thecoding sequence lacking introns).

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

“Expression control sequences”, e.g., transcriptional and translationalcontrol sequences, are regulatory sequences that flank a codingsequence, such as promoters, enhancers, suppressors, terminators, andthe like, and that provide for the expression of a coding sequence in ahost cell. In eukaryotic cells, polyadenylation signals are controlsequences. On mRNA, a ribosome binding site is an expression controlsequence.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “operatively associated with” or “under thecontrol” of transcriptional and translational control sequences in acell when RNA polymerase transcribes the coding sequence into mRNA,which is then trans-RNA spliced (if it contains introns) and translatedinto the protein encoded by the coding sequence.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface or in the membrane of acell. This sequence encodes a signal peptide, N-terminal to the maturepolypeptide, that directs the host cell to translocate the polypeptide.The term “translocation signal sequence” is used herein to refer to thissort of signal sequence.

Recombinant vectors can be introduced into host cells via calciumphosphate precipitation, infection/viral transformation,electroporation, lipofection, etc., so that many copies of the genesequence are generated. Preferably, the cloned gene is contained on ashuttle vector plasmid, which provides for expansion in a cloning cell,e.g., E. coli, and facile purification for subsequent insertion into anappropriate expression cell line, if such is desired. For example, ashuttle vector, which is a vector that can replicate in more than onetype of organism, can be prepared for replication in both E. coli andSaccharomyces cerevisiae by linking sequences from an E. coli plasmidwith sequences from the yeast 2μ plasmid.

Expression Vectors

The nucleotide sequence coding for the ER and the transcription factor,i.e., a C/EBP transcription factor, can be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. Thus, the nucleic acid encoding ER and C/EBP are eachoperatively associated with a promoter in an expression vector of theinvention. In addition, the reporter gene is operatively associated withthe HL promoter. Both cDNA and genomic sequences can be cloned andexpressed under control of such regulatory sequences. An expressionvector also preferably includes a replication origin.

A wide variety of host/expression vector combinations (i.e., expressionsystems) may be employed in expressing the DNA sequences of thisinvention. Useful expression vectors, for example, may consist ofsegments of chromosomal, non-chromosomal and synthetic DNA sequences.Suitable vectors include derivatives of SV40 and known bacterialplasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET,pGEX (Smith et al., Gene 67:31-40, 1988), pMB9 and their derivatives,plasmids such as RP4; phage DNAS, e.g., the numerous derivatives ofphage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentoussingle stranded phage DNA; yeast plasmids such as the 2μ plasmid orderivatives thereof; vectors useful in eukaryotic cells, such as vectorsuseful in insect or mammalian cells; vectors derived from combinationsof plasmids and phage DNAS, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like. Inaddition, various tumor cells lines can be used in expression systems ofthe invention.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells are used for screening or other assays, as described infra.

A preferred expression host is a eukaryotic cell (e.g., yeast, insect,or mammalian cell). More preferred is a mammalian cell, e.g., human,rat, monkey, dog, or hamster cell. In specific embodiments, infra, thecomponents of the assay system (ER, C/EBP, and reporter gene undercontrol of the HL promoter) are expressed in a hepatocarcinoma cell,specifically HepG2. Other suitable cells include, without limitation,CHO, MDCK, COS, HeLa, 3T3, and other well known cells and cell lines.Alternatively, it is possible to transfect primary cells, includingprimary stem cells, to generate a cell for an assay of the invention.

A recombinant ER or C/EBP, or both, protein may be expressedchromosomally, after integration of the coding sequence byrecombination. In this regard, any of a number of amplification systemsmay be used to achieve high levels of stable gene expression (SeeSambrook et al., 1989, supra).

Yeast expression systems can also be used according to the invention toexpress any protein of interest. For example, the non-fusion pYES2vector (XbaI, SphI, Shol, Noti, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1,and HindIII cloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI,SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloningsite, N-terminal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), to mention just two, can be employedaccording to the invention.

Expression of the protein or polypeptide may be controlled by anypromoter/enhancer element known in the art, but these regulatoryelements must be functional in the host selected for expression.Promoters which may be used to control gene expression include, but arenot limited to, cytomegalovirus (CMV) promoter (U.S. Pat. No. 5,385,839and U.S. Pat. No. 5,168,062), the SV40 early promoter region (Benoistand Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell22:787-797, 1980), the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981), the regulatorysequences of the metallothionein gene (Brinster et al., Nature296:39-42, 1982); prokaryotic expression vectors such as the β-lactamasepromoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A.75:3727-3731, 1978), or the tacpromoter (DeBoer, et al., Proc. Natl.Acad. Sci. U.S.A. 80:21-25, 1983); see also “Useful proteins fromrecombinant bacteria” in Scientific American, 242:74-94, 1980; promoterelements from yeast or other fungi such as the Gal 4 promoter, the ADC(alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter,alkaline phosphatase promoter; and transcriptional control regions thatexhibit hematopoietic tissue specificity, in particular: beta-globingene control region which is active in myeloid cells (Mogram et al.,Nature 315:338-340, 1985; Kollias et al., Cell 46:89-94, 1986),hematopoietic stem cell differentiation factor promoters, erythropoietinreceptor promoter (Maouche et al., Blood, 15:2557, 1991), etc.

Preferred vectors, particularly for cellular assays in vitro, are viralvectors, such as lentiviruses, retroviruses, herpes viruses,adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, andother recombinant viruses with desirable cellular tropism. Thus, a geneencoding a functional protein can be introduced in vitro using a viralvector or through direct introduction of DNA.

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, which entirely or almost entirely lack viralgenes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Molec. Cell. Neurosci. 2:320-330, 1991), defective herpes virusvector lacking a glyco-protein L gene (Patent Publication RD 371005 A),or other defective herpes virus vectors (International PatentPublication No. WO 94/21807, published Sep. 29, 1994; InternationalPatent Publication No. WO 92/05263, published Apr. 2, 1994); anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. (J. Clin. Invest. 90:626-630, 1992; seealso La Salle et al., Science 259:988-990, 1993); and a defectiveadeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101,1987; Samulski et al., J. Virol. 63:3822-3828, 1989; Lebkowski et al.,Mol. Cell. Biol. 8:3988-3996, 1988).

Various companies produce viral vectors commercially, including but byno means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can bemodified to efficiently deliver a nucleic acid of the invention to avariety of cell types. Various serotypes of adenovirus exist. Of theseserotypes, preference is given, within the scope of the presentinvention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5)or adenoviruses of animal origin (see WO94/26914). Those adenoviruses ofanimal origin which can be used within the scope of the presentinvention include adenoviruses of canine, bovine, murine (e.g., example:Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, andsimian (e.g., SAV) origin. Various replication defective adenovirus andminimum adenovirus vectors have been described (WO94/26914, WO95/02697,WO94/28938, WO94/28152, WO94/12649,. WO95/02697 WO96/22378).

Adeno-associated viruses. The adeno-associated viruses (AAV) are DNAviruses of relatively small size which can integrate, in a stable andsite-specific manner, into the genome of the cells which they infect.The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (see WO 91/18088; WO 93/09239; U.S. Pat.No. 4,797,368; U.S. Pat. No. 5,139,941, EP 488 528).

Retrovirus vectors. In another embodiment the gene can be introduced ina retroviral vector, e.g., as described in Anderson et al., U.S. Pat.No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat.No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al.,1988, J. Virol. 62:1120; Temin et al., U.S. Pat. No. 5,124,263; EP453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689; International Patent Publication No. WO95/07358, published Mar. 16, 1995, by Dougherty et al.; and Kuo et al.,1993, Blood 82:845. The retroviruses are integrating viruses whichinfect dividing cells. These vectors can be constructed from differenttypes of retrovirus, such as, HIV, MoMuLV (“murine Moloney leukaemiavirus”) MSV (“murine Moloney sarcoma virus”), HaSV (“Harvey sarcomavirus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcoma virus” andFriend virus. Suitable packaging cell lines have been described in theprior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719);the PsiCRIP cell line (WO 90/02806) and the GP+envAm-12 cell line (WO89/07150).

Lentivirus vectors. In another embodiment, lentiviral vectors are can beused as agents for the direct delivery and sustained expression of atransgene (for a review, see, Naldini, Curr. Opin. Biotechnol.,9:457-63, 1998; see also Zufferey, et al., J. Virol., 72:9873-80, 1998).Lentiviral packaging cell lines are available and known generally in theart (see Kim et al., J. Virology, 1998, 72:811-816). High-titerlentivirus vectors have been found to be excellent transfection agentsfor protein function assays in tissue cultured cells. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linewhich can generate virus particles at titers greater than 10⁶ IU/ml forat least 3 to 4 days (Kafri, et al., J. Virol., 73: 576-584, 1999). Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing nondividing cells in vitro.

Non-viral vectors. In another embodiment, the vector can be introducedby lipofection, as naked DNA, or with other transfection facilitatingagents (peptides, polymers, etc.). Synthetic cationic lipids can be usedto prepare liposomes for transfection of a gene (Felgner, et. al., Proc.Natl. Acad. Sci. U.S.A. 84:7413-7417, 1987; Felgner and Ringold, Science337:387-388, 1989; see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A.85:8027-8031, 1988; Ulmer et al., Science 259:1745-1748, 1993). Usefullipid compounds and compositions for transfer of nucleic acids aredescribed in International Patent Publications WO95/18863 andWO96/17823, and in U.S. Pat. No. 5,459,127. Lipids may be chemicallycoupled to other molecules for the purpose of targeting (see Mackey, et.al., supra).

Other molecules are also useful for facilitating transfection of anucleic acid, such as a cationic oligopeptide (e.g., InternationalPatent Publication WO95/21931), peptides derived from DNA bindingproteins (e.g., International Patent Publication WO96/25508), or acationic polymer (e.g., International Patent Publication WO95/21931).

It is also possible to introduce the vector as a naked DNA plasmid.Naked DNA vectors can be introduced into the desired host cells bymethods known in the art, e.g., electroporation, microinjection, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (see, e.g., Wu et al., J. Biol.Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem. 263:14621-14624,1988; Hartmut et al., Canadian Patent Application No. 2,012,311, filedMar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730,1991). Receptor-mediated DNA delivery approaches can also be used(Curiel et al., Hum. Gene Ther. 3:147-154, 1992; Wu and Wu, J. Biol.Chem. 262:4429-4432, 1987).

Screening and Chemistry

The recombinant cells of the invention that express a reporter geneunder control of the HL promoter, which in turn is regulated by an ER,provides for development of screening assays, particularly for highthroughput screening of molecules that up- or down-regulate the activityof the reporter gene expressed under the control of HL promoterexpressed after transfection or transformation of the cells.Accordingly, the present invention contemplates methods for identifyingspecific ligands of ER that modulate its ability to regulate the HLpromoter using various screening assays known in the art.

Any screening technique known in the art can be used to screen foragonists or antagonists. The present invention contemplates screens forsmall molecule ligands or ligand analogs and mimics, as well as screensfor natural ligands that bind to and agonize or antagonize the receptorin vivo. For example, natural products libraries can be screened usingassays of the invention for molecules that agonize or antagonize HLactivity via the estrogen receptor. The present invention contemplatesscreens for synthetic small molecule agents, chemical compounds,chemical complexes, and salts thereof as well as screens for naturalproducts, such as plant extracts or materials obtained from fermentationbroths. Other molecules that can be identified using the screens of theinvention include proteins and peptide fragments, peptides, nucleicacids and oligonucleotides, carbohydrates, phospholipids and other lipidderivatives, steroids and steroid derivatives, prostaglandins andrelated arachadonic acid derivatives, etc. In a specific embodiment, thetest compound can be an estrogen compound.

Knowledge of the primary sequence of the receptor, and the similarity ofthat sequence with proteins of known function, can provide an initialclue as inhibitors or antagonists. Identification and screening ofantagonists is further facilitated by determining structural features ofthe protein, e.g., using X-ray crystallography, neutron diffraction,nuclear magnetic resonance spectrometry, and other techniques forstructure determination. These techniques provide for the rationaldesign or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, Science249:386-390, 1990; Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382,1990; Devlin et al., Science, 49:404-406, 1990), very large librariescan be constructed (10⁶-10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method (Geysen et al.,Molecular Immunology 23:709-715, 1986; Geysen et al. J. ImmunologicMethod 102:259-274, 1987; and the method of Fodor et al. (Science251:767-773, 1991) are examples. Furka et al. (14th InternationalCongress of Biochemistry, Volume #5, Abstract FR:013, 1988; Furka, Int.J. Peptide Protein Res. 37:487-493, 1991), Houghton (U.S. Pat. No.4,631,211, issued December 1986) and Rutter et al. (U.S. Pat. No.5,010,175, issued Apr. 23, 1991) describe methods to produce a mixtureof peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., Proc. Natl.Acad. Sci. USA 90:10700-4, 1993; Ohlmeyer et al., Proc. Natl. Acad. Sci.USA 90:10922-10926, 1993; Lam et al., International Patent PublicationNo. WO 92/00252; Kocis et al., International Patent Publication No. WO9428028) and the like can be used to screen for NF-E4 ligands accordingto the present invention.

Test compounds are screened from large libraries of synthetic or naturalcompounds. Numerous means are currently used for random and directedsynthesis of saccharide, peptide, and nucleic acid based compounds.Synthetic compound libraries are commercially available from MaybridgeChemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.),Brandon Associates (Merrimack, N.H.), and Microsource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from e.g. PanLaboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readilyproducible. Additionally, natural and synthetically produced librariesand compounds are readily modified through conventional chemical,physical, and biochemical means (Blondelle et al., Tib Tech, 14:60,1996).

In Vitro Screening Methods

According to the present invention, a recombinant ER-HL promoteractivity system is constructed. Candidate agents are added to in vitrocell cultures of host cells, prepared by known methods in the art, andthe activity of the reporter gene is measured. Various in vitro systemscan be used to analyze the effects of a new compound on reporter geneexpression under control of the HL promoter. Preferably, each experimentis performed in triplicate at multiple different dilutions of compound.

Reporter genes for use in the invention encode detectable proteins,include, but are by no means limited to, chloramphenicol transferase(CAT), β-galactosidase (β-gal), luciferase, green fluorescent protein(GFP) and derivatives thereof, yellow fluorescent protein andderivatives thereof, alkaline phosphatase, other enzymes that can beadapted to produce a detectable product, and other gene products thatcan be detected, e.g., immunologically (by immunoassay).

GFP has been modified to produce proteins that remain functional buthave different fluorescent properties. Heim et al (U.S. Pat. No.5,625,048) modified GFP resulting in amino-acid changes which exhibiteddifferent excitation and emission spectra with visibly distinct colorsand increased intensities of emission. Bjorn et al (WO 96/23898)developed a new construct which encoded a modified GFP but alsocontained an enzyme recognition site. Bjorn et al (WO 97/11094) alsodeveloped new fluorescent proteins with increased intensity compared tothe parent proteins. Hauswirth et al (WO 97/266333) developed a GFPprotein optimized to provide higher levels of expression in mammaliancells. Gaitanaris et al (WO 97/42320) modified GFP resulting to increasethe intensity of fluorescence, e.g., by some twenty times greater thanwild-type GFP, therefore increasing the sensitivity of detection. Cubittet al (WO 98/06737) developed modified GFP which could be easilydistinguished from the already known green and blue fluorescentproteins. Evans et al (WO 98/21355) developed new GFP mutants excitablewith blue and white light.

The host cell screening system of the invention permits two kinds ofassays: direct activation assays (agonist screen) and inhibition assays(antagonist screen). An agonist screen involves detecting changes in thelevel of expression of the reporter gene by the host cell contacted witha test compound; generally, reporter gene expression decreases. If thereporter gene is expressed, the test compound has not affected the HLpromoter via the ER; if the reporter gene expression decreases, the testcompound is a candidate for developing an HL suppressive drug.

An antagonist screen involves detecting changes in the level ofexpression of the reporter gene by the host cell contacted with a testcompound; generally, reporter gene expression is not affected orincreases. If in the presence of a known ER agonist the test compounddoes not prevent inhibition of HL activity or increases the observedagonist HL inhibition, the test compound may not recognize the ERisoform or may be producing effects on HL activity through mechanismsother then interaction with the ER.

The reporter gene assay system described here may be used in ahigh-throughput primary screen for agonists and antagonists, or it maybe used as a secondary functional screen for candidate compoundsidentified by a different primary screen, e.g., a binding assay screenthat identifies compounds that interact with the receptor and affect HLpromoter activity.

High-Throughput Screen

Agents according to the invention may be identified by screening inhigh-throughput assays, including without limitation cell-based orcell-free assays. It will be appreciated by those skilled in the artthat different types of assays can be used to detect different types ofagents. Several methods of automated assays have been developed inrecent years so as to permit screening of tens of thousands of compoundsin a short period of time (see, e.g., U.S. Pat. Nos. 5,585,277;5,679,582; and 6,020,141). Such high-throughput screening methods areparticularly preferred. Alternatively, simple reporter-gene based cellassays such as the one described here are also highly desirable. The useof high-throughput screening assays to test for agents is greatlyfacilitated by the availability of large amounts of purifiedpolypeptides, as provided by the invention.

Estrogen Compounds

An “estrogen compound” is defined as any of the structures described inthe 11th edition of “Steroids” from Steraloids Inc., Wilton N. H., hereincorporated by reference. Included in this definition are non-steroidalestrogens described in the aforementioned reference. Other estrogencompounds included in this definition are estrogen derivatives, estrogenmetabolites, estrogen precursors, and selective estrogen receptormodulators (SERMs). The term also encompasses molecules thatspecifically trigger the estrogen effect described herein of regulatingHL promoter activity. Also included are mixtures of more than oneestrogen or estrogen compound. Examples of such mixtures are provided inTable II of U.S. Pat. No. 5,554,601 (see column 6). Examples ofestrogens having utility either alone or in combination with otheragents are provided, e.g., in U.S. Pat. No. 5,554,601. In anotherembodiment, the estrogen compound is a composition of conjugated equineestrogens (PREMARIN™; Wyeth-Ayerst).

β-estrogen is the β-isomer of estrogen compounds. α-estrogen is theα-isomer of estrogen components. The term “estradiol” is either α- orβ-estradiol unless specifically identified.

The term “E2,” is synonymous with β-estradiol, 17β-estradiol, and β-E2.αE2 and α-estradiol is the α isomer of βE2 estradiol.

Preferably, a non-feminizing estrogen compound is used. Such a compoundhas the advantage of not causing uterine hypertrophy and otherundesirable side effects, and thus, can be used at a higher effectivedosage. Examples of non-feminizing estrogen include Raloxifene (Evista;Eli Lilly), Tamoxifen (Nolvadex; Astra Zeneca), and other selectiveestrogen receptor modulators.

In addition, certain compounds, such as the androgen testosterone, canbe converted to estradiol in vivo.

EXAMPLE

The present invention will be better understood by reference to thefollowing Examples, which are provided as exemplary of the invention,and not by way of limitation.

Example 1 Transformed HepG2 Cell Assay System for Compounds That InhibitHepatic Lipase Promoter by Binding the Estrogen Receptor

Materials and Methods

Human hepatocarcinoma cells (HepG2, ATTC# HB8065), were grown a 75 cm²flask in Dulbecco's modified Eagle medium (DMEM) (Gibco BRL; Rockville,Md.), supplemented with 1%, by volume, glutamax; 1%, by volume,penicillin/strepomycin; 1%, by volume, non-essential amino acids; and10%, by volume, heat inactivated fetal bovine serum. Cell media wasreplaced every 2-3 days. Confluent cells were rinsed once withphosphate-buffered saline and 5 mL of a prewarmed solution comprised ofabout 0.05% trypsin, by volume, and about 0.5 mM ethylenediaminetetraacetic acid was added. Cells were left at room temperature for 5minutes and cell dislodged from flask by tapping flask 50 times.Trypsinization was halted by the addition of about 8.5 mL of growth cellculture media. Cells were transferred to a test tube and centrifuged atabout 300×g for about 5 minutes. Total cells were counted andresuspended at a concentration of about 1×10⁷ cells/200 μL of growthcell culture media.

Resuspended cells were supplemented with 50 μg of hepatic lipasepromoter plasmid (−1557 to +43), subcloned into the luciferase pGL2reporter vector (Promega); 25 μg human estrogen receptor, which wassubcloned into a pCDNA3 vector (Invitrogen); 25 μg CAAT enhancer bindingprotein (C/EBPα) expression vector, and 20 μg β-galactosidase reporterplasmid (pCH110, Pharmacia). The mixture was transferred to a 0.2 mL BTXdisposable cuvetter (P/N 620). Cells were electroporated using a BTX ECM600 (San Diego, Calif.), at 100 V, 1700 VF and 72 Ω. Cells were left atroom temperature for about 20 minutes. Approximately 20.5 mL of growthcell culture media was added to the cells. Cells were then seeded atabout 200 μL per well of a 96 well plate. Cells were then incubated atabout 37° C. for 4 hours.

Media was aspirated and replaced with 200 μL deficient media, which wascomprised of Phenol Red Free-DMEM (Gibco BRL) supplemented with 1%, byvolume, glutamax; 1%, by volume, penicillin/strepomycin; 1%, by volume,non-essential amino acids; and 10%, by volume, heat inactivated, andcharcoal-stripped fetal bovine serum, containing varying concentrationsof estrogen test compounds. Cells were incubated at about 37° C. for20-24 hours.

After estrogen treatment, cells were washed with phosphate-bufferedsaline and lysed with Cell Culture Lysis Reagent (Promega; Madison,Wis.), 50 μL/well, by shaking the plate at room temperature for about 20minutes. From each well, 35 μL of cell lysate was were transferred to a96 lumat plate. Luciferase activity was determined by addition of 100 μLluciferase substrate and emitted light was detected with a DentateMicrofluor WHT FB using the Luciferase Assay System (Promega; Madison,Wis.). β-galactosidase activity was determined by adding 10 μL of celllysate and 100 μL β-galactosidase light emission buffer (Tropix; BedfordMass.) to a lumat plate. β-galactosidase activity was determined with aMicrolumat LB 96P (EG & G Berthold). Emitted light, for each assay, wasdetected for 10 seconds.

Data were collected and analyzed with the JMP statistical program.Background RLU values were subtracted from both the luciferase and β-galvalues. The data were normalized by division of the corrected luciferasevalues by the corrected β-gal values. Dose-response curves weregenerated for dose (X-axis) versus % activity (Y-axis) by analysis ofdata by log transformation and fitted by Huber weighting to provideefficacy (% maximal value) and potency values (EC₅₀) for comparison to17-β estradiol. Mean and standard deviations were determined from atleast two separate experiments with an n=4 for each experiment.

Transfected cells in multi-well plates were treated with test compoundslisted in Table 1 for 16-24 hours. Luciferase activity was determined incell lysates. Under these conditions, 17β-estradiol inhibited HLpromoter activity by about 75%. Repression of HL promoter activity is ERand ligand dependent. Results are summarized in Table 1. TABLE 1 HepaticLipase Inhibition Compound Name Efficacy^(a) (%) IC50 (nM) 17β-estradiol100  88 estrone 95 26 17αΔ8,9 dehydroestradiol — —17β-Δ8,9-dehydroestradiol 88 12 Δ8,9-dehydroestrone 93 10 equilenin 8238 17α-dyhydroequilenin — — 17β-dyhydroequilenin — — equlin — —17α-dihydroequilin 89 164  17β-dihydroequilin 149  469  17α-estradiol 69457  estradiene — — raloxifene —^(a)Efficacy is based upon a value of 100% effect of 17β-estradiolinhibition of HL activity.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that values are approximate, and areprovided for description.

Patents, patent applications, publications, procedures, and the like arecited throughout this application, the disclosures of which areincorporated herein by reference in their entireties.

1. An isolated cell comprising: (i) a first exogenous nucleic acidmolecule which encodes an estrogen receptor; (ii) a second exogenousnucleic acid molecule which encodes a chicken ovalbumin upstreampromoter-transcription factor (COUP-TF); and (iii) a reporteroperatively associated with a hepatic lipase (HL) promoter.
 2. The cellof claim 1, wherein the the estrogen receptor is a human estrogenreceptor.
 3. The cell of claim 1, wherein the estrogen receptor is anERα.
 4. The cell of claim 1, wherein the COUP-TF is selected from thegroup consisting of ARP-1, EAR-2, and EAR-3.
 5. The cell of claim 1,wherein the estrogen receptor, the COUP-TF, and the reporter operativelyassociated with a hepatic lipase promoter are expressed from separatevectors, or the same vector.
 6. The cell of claim 1, wherein the hepaticlipase promoter is positioned proximal to the 5′ end of human hepaticlipase coding region.
 7. The cell of claim 1, wherein the hepatic lipasepromoter comprises the human hepatic lipase promoter region from −1557to +43, relative to the human hepatic lipase coding region start site.8. The cell of claim 1, wherein the reporter encodes a protein selectedfrom the group consisting of luciferase, green fluorescent protein,yellow fluorescent protein, β-galactosidase, chloramphenicoltransferase, horseradish peroxidase, and alkaline phosphatase.
 9. Thecell of claim 1, wherein the cell is selected from the group consistingof a yeast cell, an insect cell, and a mammalian cell.
 10. The cell ofclaim 9, wherein the cell is a mammalian cell, and the mammalian cell isselected from the group consisting of a human cell, a rat cell, a monkeycell, a dog cell, and a hamster cell.
 11. The cell of claim 1, whereinthe cell is a hepatocarcinoma cell.
 12. The cell of claim 1, wherein thecell is selected from the group consisting of a HepG2 cell, a COS cell,a CHO cell, a MDCK cell, a Hela cell, a 3T3 cell, and a primary cell.13. The cell of claim 1, wherein the first and second exogenous nucleicacid molecules are inserted into expression vectors.
 14. The cell ofclaim 12, wherein the expression vector is selected from the groupconsisting of pCR1, pBR322, pMal-C2, pET, pGEX, pMB9, RP4, pYES2,pYESHisA, pYESHisB, pYES HisC, pcDNA3, and a viral vector.
 15. An assaysystem for compounds that modulate hepatic lipase promoter activitycomprising a population of cells of claim 1, wherein the number of cellsin a single assay system is sufficient to express a detectable amount ofthe protein encoded by the reporter under conditions of maximum reporterexpression.
 16. A method for identifying a compound that regulates an HLpromoter, which method comprises detecting a change in the level ofexpression of a reporter in an assay system of claim 15 contacted with atest compound, wherein detection of a change in the level of expressionof the reporter indicates that the test compound regulates the HLpromoter.
 17. The method according to claim 16, wherein the testcompound is an estrogen or an estrogen analog.
 18. The method accordingto claim 16, wherein the level of reporter expression decreases whencontacted with a test compound that regulates the HL promoter.
 19. Amethod of increasing the levels of HDL in a patient, comprisingadministering the patient in need thereof with an effective amount of anestrogen compound selected from the group consisting of 17β-estradiol,estrone, 17αΔ8,9 dehydroestradiol, 17β-Δ8,9-dehydroestradiol,Δ8,9-dehydroestrone, equilenin, 17α-dyhydroequilenin,17β-dyhydroequilenin, equlin, 17α-dihydroequilin, 17β-dihydroequilin,17α-estradiol, estradiene, tamoxifen, and raloxifene, thereby increasingthe levels of HDL in the patient.
 20. A method of decreasing the levelsof LDL in a patient, comprising administering the patient in needthereof with an effective amount of an estrogen compound selected fromthe group consisting of 17β-estradiol, estrone, 17αΔ8,9dehydroestradiol, 17β-α8,9-dehydroestradiol, Δ8,9-dehydroestrone,equilenin, 17α-dyhydroequilenin, 17β-dyhydroequilenin, equlin,17α-dihydroequilin, 17β-dihydroequilin, 17α-estradiol, estradiene,tamoxifen, and raloxifene, thereby decreasing the levels of LDL in thepatient.
 21. An isolated cell comprising: (i) a first exogenous nucleicacid molecule which encodes an estrogen receptor; (ii) a secondexogenous nucleic acid molecule which encodes a CCAAT/enhancer-bindingprotein (C/EBP) transcription factor; and (iii) a reporter operativelyassociated with a hepatic lipase (HL) promoter, wherein the first and/orsecond exogenous nucleic acid molecule is/are inserted into a viralvector.
 22. The cell of claim 21, wherein the viral vector is selectedfrom the group consisting of a lentivirus vector, a retrovirus vector, aherpes virus vector, an adenovirus vector, an adeno-associated virusvector, a vaccinia virus vector, and a baculovirus vector.